Chemical insecticides are an integral component of modern agriculture, and are an effective means for reducing crop damage by controlling insect pests. However, chemical agents are under continuous scrutiny due to the potential for environmental contamination, selection of resistant populations of agronomic pests, and toxicity to non-target organisms such as beneficial insects, aquatic organisms, animals and man. As a result, alternative strategies for insect control are being sought that are effective and yet benign to non-target populations and the environment.
One of these strategies is to use microorganisms that are naturally occurring pathogens of target insect populations. However, many candidate entomopathogens that would be promising insect control agents lack the properties of classical chemical insecticides such as host-specificity and rapid action that farmers and others in agribusiness have grown accustomed to. Viruses from the family Baculoviridae are host-specific and have inert environmental properties, but lack the ability to rapidly neutralize a target population before significant crop damage takes place. Fortunately, modern molecular biology provides the tools to produce recombinant baculoviruses engineered for use as biological control agents.
An attractive attribute of baculoviruses is their narrow host specificity. These viruses infect only arthropods and possess relatively narrow host ranges even within a particular insect order. Host specificity has been examined by electron microscopy, DNA hybridization and recombinant DNA technology (References 1-3). These studies indicate that the narrow host range is due, at least in part, to the inability of baculoviruses to transfer viral DNA into the mammalian cell nucleus.
Baculoviruses are divided into three subfamilies, including non-occluded baculoviruses (NOVs), granulosis viruses (GVs) and nuclear polyhedrosis viruses (NPVs). Although certain GVs and NOVs have been carefully studied, NPVs are the most thoroughly characterized of the baculovirus subfamilies. Examples of NPVs include Autographa californica NPV, Spodoptera exigua NPV, Heliothis armigera NPV, Helicoverpa zea NPV, Spodoptera frugiperda NPV, Trichoplusia ni NPV, Mamestra brassicae NPV, Lymantria dispar NPV, Spodoptera litturalis NPV, Syngrapha facifera NPV, Choristoneura fumiferana NPV, Anticarsia gemmatalis NPV, and Heliothis virescens NPV.
Due in part to the availability of efficient cell culture systems and facile cloning vectors, NPVs have been used as eukaryotic expression vectors to synthesize desirable heterologous proteins (4,5). One virus in particular, Autographa californica NPV (AcNPV), is the accepted model virus for introduction and expression of heterologous genes in baculovirus expression systems. Although this virus is routinely used as an important in vitro means of providing for high yields of recombinant proteins in a eukaryotic expression system, thus affording appropriate post-translational modification of expressed proteins, AcNPV is capable of infecting many families of Lepidopteran insects that are important economic pests.
In spite of the potential practical advantages of baculovirus-based pest control agents, a variety of disadvantages have curtailed their use in modem agriculture. The most significant barrier to more widespread use of these viruses in row-crop agriculture is the significant time delay between their application and effective control of crop damage caused by the host insects. Unlike the rapid effects observed upon application of classical chemical insecticides, significant wild-type baculovirus-mediated insect control occurs only after in vivo populations of virus have reached levels high enough to compromise host activity. This may occur as long as several weeks after infection in a cycle that involves two types of virions. Following infection of insect cells, budded virions (BVs or extracellular virus, ECV) are produced upon movement of nucleocapsids to the plasma membrane. These virions shed their nuclear-derived coat in the cytoplasm and bud through the cytoplasmic membrane into the hemocoel of the insect host. This process leads to systemic infection of the host insect. Later in the infection process, virions become occluded (occluded virions) by a protein matrix consisting substantially of the polyhedrin protein, thus forming polyhedral inclusion bodies (PIBs or occlusion bodies, OBs). These inclusion bodies are the orally infectious form of the virus and provide for horizontal transmission of the virus between insect hosts (6,7). Uninfected larvae feed on virus-contaminated substrates and ingest PIBs. The proteinaceous matrix is solubilized by the action of the basic pH of the insect midgut found in many lepidopterous larvae. The liberated virion nucleocapsids, containing the viral DNA genome, attach to and infect the epithelial cells of the larval midgut. Typically, the infected insect will continue to develop and consume plant material while the virus exponentially propagates within the host. Eventually, often after several weeks or longer have passed, the infected larvae will become fully involved and expire.
Through the use of recombinant DNA technology, NPVs have been genetically engineered to increase their rate of insect killing by either the introduction of genes directing the expression of insecticidal proteins or the deletion of genes from the viral genome (8-10). Both strategies yield biological insecticides that display more rapid insect control than wild-type, unengineered NPVs. The most effective recombinant NPVs have been engineered to express insect-selective neurotoxins (11-18). The expressed toxins accelerate the onset of cytotoxicity, resulting in more rapid insect control. These recombinant viruses kill their hosts in 20-30% less time than wild-type NPVs.
Baculoviruses destined for use as biological pest control agents must be produced in large quantities. Mass-production of virus can be accomplished with standard in vitro insect cell culture systems or by in vivo production in infected insect larvae. Yields of viral particles in either system are dependent on sufficient viral replication, which is in turn dependent on maintenance of healthy cells or insects. Premature cell cytotoxicity or insect death will necessarily limit viral replication, thereby reducing the number of progeny virus produced.
Genetically modified baculoviruses, engineered to more rapidly neutralize target insects, may also result in less viral replication and result in lower yields of viral progeny per infected cell (in vitro) or per infected insect (in vivo). Thus, the means used to improve the efficacy of baculovirus-mediated insect control agents, making them viable alternatives or adjuncts to traditional chemical insecticides, may in fact defeat the economic viability of this pest control strategy. Accordingly, a method for efficient production of insecticidal baculoviruses is needed that overcomes the barrier of premature cytotoxicity or premature killing of the host cell or insect.